Quantifying Tau Phosphorylation Levels

Levels of total and phosphorylated tau were assessed using the Low-tau ELISA protocol previously published [1 (link), 22 (link)]. 96-well plates were coated for 48 h at 4 °C with specific purified monoclonal tau antibodies (DA31, CP13, PHF1, RZ3) at a concentration of 6 μg/ml. After washing, plates were blocked for 1 h at RT using StartingBlock buffer (Thermo Fisher Scientific, Waltham, MA). Brain samples and standards were diluted in 20% SuperBlock buffer (Thermo Fisher Scientific) in 1XTBS and loaded on the plates. Once the samples were added, the total tau detection antibody DA9-HRP, diluted 1:50 in 20% SuperBlock in 1XTBS, was added to the samples and tapped to combine. Plates were then incubated overnight at 4 °C. Next day, 1-Step ULTRA TMB-ELISA (Thermo Fisher Scientific) was added for 30 min at RT, followed by 2 M H₂SO₄ to stop the reaction. Plates were read with Infinite m200 plate reader (Tecan, San Jose, CA) at 450 nm.